Brian Hall

An Essential Role for the NLRP3 Inflammasome in Host Defense against the Human Fungal Pathogen Candida albicans

Candida albicans is an opportunistic fungal pathogen causing life-threatening mucosal and systemic infections in immunocompromised humans. Using a murine model of mucosal Candida infection, we investigated the role of the proinflammatory cytokine IL-1beta in host defense to Candida albicans. We find that the synthesis, processing, and release of IL-1beta in response to Candida are tightly controlled and first require transcriptional induction, followed by a second signal leading to caspase-1-mediated cleavage of the pro-IL-1beta cytokine. The known fungal pattern recognition receptors TLR2 and Dectin-1 regulate IL-1beta gene transcription, whereas the NLRP3-containing proinflammatory multiprotein complex, the NLRP3 inflammasome, controls caspase-1-mediated cleavage of pro-IL-1beta. Furthermore, we show that TLR2, Dectin-1, and NLRP3 are essential for defense against dissemination of mucosal infection and mortality in vivo. Therefore, in addition to sensing bacterial and viral pathogens, the NLRP3 inflammasome senses fungal pathogens and is critical in host defense against Candida.

A Novel Role for the NLRC4 Inflammasome in Mucosal Defenses against the Fungal Pathogen Candida albicans

Candida sp. are opportunistic fungal pathogens that colonize the skin and oral cavity and, when overgrown under permissive conditions, cause inflammation and disease. Previously, we identified a central role for the NLRP3 inflammasome in regulating IL-1β production and resistance to dissemination from oral infection with Candida albicans. Here we show that mucosal expression of NLRP3 and NLRC4 is induced by Candida infection, and up-regulation of these molecules is impaired in NLRP3 and NLRC4 deficient mice. Additionally, we reveal a role for the NLRC4 inflammasome in anti-fungal defenses. NLRC4 is important for control of mucosal Candida infection and impacts inflammatory cell recruitment to infected tissues, as well as protects against systemic dissemination of infection. Deficiency in either NLRC4 or NLRP3 results in severely attenuated pro-inflammatory and antimicrobial peptide responses in the oral cavity. Using bone marrow chimeric mouse models, we show that, in contrast to NLRP3 which limits the severity of infection when present in either the hematopoietic or stromal compartments, NLRC4 plays an important role in limiting mucosal candidiasis when functioning at the level of the mucosal stroma. Collectively, these studies reveal the tissue specific roles of the NLRP3 and NLRC4 inflammasome in innate immune responses against mucosal Candida infection.

Quantitative image based apoptotic index measurement using multispectral imaging flow cytometry: a comparison with standard photometric methods

Morphological characterization by microscopy remains the gold standard for accurately identifying apoptotic cells using characteristics such as nuclear condensation, nuclear fragmentation, and membrane blebbing. However, quantitative measurement of apoptotic morphology using microscopy can be time consuming and can lack objectivity and reproducibility, making it difficult to identify subtle changes in large populations. Thus the apoptotic index of a sample is commonly measured by flow cytometry using a variety of fluorescence intensity based (photometric) assays which target hallmarks of apoptosis with secondary markers such as the TUNEL (Terminal Deoxynucleotide Transferase dUTP Nick End Labeling) assay for detection of DNA fragmentation, the Annexin V assay for surface phosphatidylserine (PS) exposure, and fluorogenic caspase substrates to detect caspase activation. Here a novel method is presented for accurate quantitation of apoptosis based on nuclear condensation, nuclear fragmentation, and membrane blebbing using automated image analysis on large numbers of images collected in flow by the ImageStream multispectral imaging cytometer. Additionally the measurement of nuclear fragmentation correlates with the secondary methods of detection of apoptosis over time, indicating that it is also an early marker for apoptosis. False-positive and false-negative events associated with each photometric flow cytometry based method are quantitated and can be automatically removed/included where appropriate. Acquisition of multi-spectral imagery on large numbers of cells couples the quantitative advantage of flow cytometry with the accuracy of morphology-based algorithms allowing more complete and robust analysis of apoptosis.

β-Defensin 1 plays a role in acute mucosal defense against Candida albicans.

Candida is an opportunistic fungal pathogen that colonizes the mucosal tract of humans. Pathogenic infection occurs in the presence of conditions causing perturbations to the commensal microbiota or host immunity. Early innate immune responses by the epithelium, including antimicrobial peptides (AMPs) and cytokines, are critical for protection against overgrowth. Reduced salivary AMP levels are associated with oral Candida infection, and certain AMPs, including human β-defensins 1–3, have direct fungicidal activity. In this study, we demonstrate that murine β-defensin 1 (mBD1) is important for control of early mucosal Candida infection and plays a critical role in the induction of innate inflammatory mediators. Mice deficient in mBD1, as compared with wild-type mice, exhibit elevated oral and systemic fungal burdens. Neutrophil infiltration to the sites of mucosal Candida invasion, an important step in limiting fungal infection, is significantly reduced in mBD1-deficient mice. These mice also exhibit defects in the expression of other AMPs, including mBD2 and mBD4, which may have direct anti-Candida activity. We also show that mBD1 deficiency impacts the production of important antifungal inflammatory mediators, including IL-1β, IL-6, KC, and IL-17. Collectively, these studies demonstrate a role for the mBD1 peptide in early control of Candida infection in a murine model of mucosal candidiasis, as well as in the modulation of host immunity through augmentation of leukocyte infiltration and inflammatory gene regulation.

CD209(+) macrophages mediate host defense against Propionibacterium acnes.

Propionibacterium acnes is a major etiological factor of acne, triggering an inflammatory response in part through the activation of TLR2. In this study, we demonstrate that activation of peripheral blood monocytes with P. acnes in vitro induced their differentiation into two distinct innate immune cell subsets, CD209+ macrophages and CD1b+ dendritic cells. Furthermore, P. acnes induced expression of mRNA for the cytokines IL-15 and GM-CSF, which differentiate CD209+ and CD1b+ cells, respectively. The CD209+ cells were more effective in uptake of P. acnes, compared with the CD1b+ cells, and demonstrated a 2-fold greater antimicrobial activity against the phagocytosed bacteria. Although CD1b+ cells secreted inflammatory cytokines in response to both P. acnes and a TLR2 ligand control, the CD209+ cells responded only to P. acnes. The addition of all-trans retinoic acid, a commonly used agent for the treatment of acne, directly induced differentiation of monocytes into CD209+ macrophages and enhanced the P. acnes-mediated differentiation of the CD209+ subset. Therefore, the differentiation of monocytes into CD209+ macrophages and CD1b+ dendritic cells distinctly mediate the innate immune response to P. acnes.

Cutting Edge: Critical Role of Glycolysis in Human Plasmacytoid Dendritic Cell Antiviral Responses

Plasmacytoid dendritic cells (pDCs) are vital to antiviral defense, directing immune responses via secretion of huge concentrations of IFN-α. These cells are critical in protecting the lung against clinically relevant respiratory viruses, particularly influenza (Flu), a virus responsible for substantial worldwide morbidity and mortality. How pDC responses to such viral pathogens are regulated, however, is poorly understood in humans. Using an unbiased approach of gene chip analysis, we discovered that Flu significantly affects metabolism in primary human pDCs. We demonstrate that Flu and RV, another common respiratory virus, induce glycolysis in pDCs and that this metabolic pathway regulates pDC antiviral functions, including IFN-α production and phenotypic maturation. Intranasal vaccination of human volunteers with live influenza virus also increases glycolysis in circulating pDCs, highlighting a previously unrecognized potential role for metabolism in regulating pDC immune responses to viral infections in humans.